Syngeneic Models. Progress immunotherapeutic development with CrownBio s panel of over 30 syngeneic models

Transcription

1 Syngeneic Models Progress immunotherapeutic development with CrownBio s panel of over 3 syngeneic models Discover the benefits of using our fully characterized and checkpoint inhibitor benchmarked syngeneic models to accelerate your immuno-oncology drug discovery programs. The development of novel immunotherapeutics presents many challenges, including the need for immunocompetent preclinical models. Syngeneic mouse models are undergoing a resurgence as an accessible platform to evaluate the efficacy and MOA of novel agents and combination strategies. CrownBio provides an extensive syngeneic platform of over 3 models covering more than 15 cancer types for immunotherapeutic assessment. Select the most appropriate models for checkpoint inhibitor novel agent/combination studies using a vast array of benchmarking data (e.g. anti-pd-1, PD-L1, CTLA-4 antibodies) complemented by immunoprofiling and NGS. Choose the right model for bacterial, viral, and vaccine immunotherapy research based on baseline immunophenotyping at the subcutaneous or orthotopic site. Evaluate efficacy quickly with standard subcutaneous models, alongside disease relevant tumor microenviroment in orthotopic and metastatic sites with bioluminescent imaging. Assess immunomodulatory effects through post-treatment immunoprofiling including T cell infiltration. Fast track new immuno-oncology agents using the first large-scale in vivo syngeneic screening platform. Historical origins of in vitro cell line: murine tumor Implant into original inbred mouse strain Syngeneic Models for I/O Research Standard subcutaneous models for I/O agent evaluation Advanced orthotopic, bioluminescent, and metastic modeling Checkpoint inhibitor benchmarking Combination therapy strategies Immune cell profiling Large scale in vivo screening

2 Syngeneic Models Key Facts CrownBio provides a well characterized Syngeneic Model Panel: Over 3 models covering 15 cancer types, with further models undergoing validation. Standard subcutaneous models for efficacy evaluation, complemented by orthotopic models to better recapitulate the tumor microenvironment, and metastatic models allowing targeting of clinically relevant metastatic invasion. Bioluminescent metastatic models to monitor in-life disease progression, and primary to end stage disease. Full validation data (baseline and post-treatment immunoprofiling, immunotherapy, standard of care, and NGS data) easily searchable through MuBase, CrownBio s online collated immuno-oncology model database. Checkpoint inhibitor benchmarking data including anti-pd-1, PD-L1, and CTLA-4 antibodies to select the appropriate models for single agent and combination studies, including combination immunotherapy and immunotherapy + chemotherapy (including inducer of ICD) strategies. Validated immunoprofiling including treatment induced T cell infiltration assessment to characterize immunomodulatory effects of novel agents and treatment regimens. Microbiome analysis to correlate gut microbiomes across our syngeneic models with response to therapy. CrownBio s large-scale, in vivo syngeneic screening platform MuScreen, the first screening platform of its type, to fast track immunotherapy compounds. Syngeneic Model Use in Preclinical Immuno- Oncology Research Evaluating immunotherapeutic agents brings many challenges, including the need for preclinical models within immunocompetent hosts. Syngeneic mouse models have seen a resurgence in use as a straightforward platform enabling efficacy testing and elucidation of the mechanism of action of new immuno-oncology treatments. Syngeneic mouse tumors are allografts derived from immortalized mouse cancer cell lines which originate from the same inbred strain of mice. The recipient mice have fully competent mouse immunity and are histocompatible to the allografted tumors. Models have now been extensively profiled genomically and immunologically (both pre- and post-treatment), and for agent efficacy to allow simple and rapid model selection for preclinical studies. Agents commonly tested using syngeneics include checkpoint inhibitors such as anti-pd-1 and PD-L1 antibodies in proof of concept studies. Syngeneics can also be utilized for evaluating a wide range of other immunotherapeutics, including bacterial, viral, and vaccine therapies, all of which have driven syngeneics to become one of the most commonly utilized immuno-oncology models in preclinical investigations. CrownBio Provides a Large and Well Profiled Panel of Syngeneic Models Our large panel of well validated syngeneic models covers over 15 cancer types and more than 3 individual models (summarized in Table 1, availability site by site is covered within our In Vivo Cancer Pharmacology Model catalogs available on request). CrownBio are constantly improving and expanding the syngeneic collection, and our pipeline of models currently undergoing validation includes: breast C127I model chondrogenic ATDC5 model colon CMT-93 model liver Hepa1c1c7 model lung LA-4 model kidney RAG model.

3 Table 1: Summary of Syngeneic Immunotherapy Models X = data available; *bioluminescent models established with further validation ongoing. Cancer Type Cell Line Model Type Anti-PD-1 Anti-PD-L1 Anti-CTLA-4 RNAseq Immune Cell Profiling Bladder MBT-2* Subcutaneous X X X X X Breast 4T1* Subcutaneous, orthotopic, metastatic, bioluminescent X (s.c., ortho) X (s.c., ortho) X (s.c., ortho*) X (s.c., ortho*) X (ortho) Ongoing (s.c.) EMT6* Subcutaneous, orthotopic, bioluminescent X (s.c.) X (s.c.) X (s.c.) X (s.c.) X (s.c.) JC Subcutaneous Ongoing Ongoing Ongoing Ongoing X Colon Colon26 Subcutaneous X X X Ongoing X CT-26.WT* Subcutaneous X X X X X Fibrosarcoma WEHI-164 Subcutaneous X Ongoing Ongoing Ongoing Ongoing Glioma GL261 Subcutaneous, orthotopic Ongoing (s.c., ortho) Ongoing (s.c., ortho) Ongoing (s.c., ortho) X (s.c.) Ongoing (ortho) Ongoing (s.c., ortho) Kidney Renca* Subcutaneous X X X X X Leukemia C1498 Subcutaneous Ongoing Ongoing Ongoing Ongoing X L121 Subcutaneous X X X X X Liver H22* Subcutaneous, orthotopic, bioluminescent X X X X X Hepa 1-6* Subcutaneous, orthotopic, bioluminescent X (s.c.) X (s.c.) X (s.c.) Ongoing (s.c.) X (s.c.) Lung KLN25 Subcutaneous X X X X X LL/2 (LLC1)* Subcutaneous, metastatic X X X X X Lymphoma A2 Subcutaneous X X X X X E.G7-OVA Subcutaneous X Ongoing Ongoing Ongoing Ongoing EL4 Subcutaneous X X X X X L5178-R (LY-R) Subcutaneous X X X Ongoing X P388D1 Subcutaneous X X X X X Mastocytoma P815* Subcutaneous Ongoing X Ongoing Ongoing Ongoing Melanoma B16-BL6 Subcutaneous X X X X X B16-F Subcutaneous Ongoing Ongoing Ongoing Ongoing Ongoing B16-F1 Subcutaneous Ongoing Ongoing Ongoing Ongoing Ongoing B16-F1* Subcutaneous, metastatic, bioluminescent X X X X X Clone M-3 (Cloudman S91) Subcutaneous Ongoing X Ongoing Ongoing Ongoing Myeloma J558 Subcutaneous Ongoing Ongoing Ongoing Ongoing X MPC-11 Subcutaneous X X X X X P3X63Ag8U.1 Subcutaneous Ongoing Ongoing Ongoing Ongoing Ongoing Neuroblastoma N1E-115 Subcutaneous Ongoing Ongoing Ongoing Ongoing Ongoing Neuro-2a Subcutaneous Ongoing Ongoing Ongoing Ongoing Ongoing Pancreatic Pan2* Subcutaneous, orthotopic, bioluminescent X (s.c.) X (s.c.) X (s.c.) X (s.c.) X (s.c.) Prostate RM-1* Subcutaneous X X X X X Standard Subcutaneous Models to Evaluate Novel Immunotherapies CrownBio standard syngeneic models shown in Table 1 are fully validated with growth, standard of care (SoC) and/or immunotherapy treatment data available. Complete background information, growth, and treatment data on models are included within MuBase our easy to use, proprietary online database. Models can be quickly searched and compared to find those appropriate for indivdual studies. Models with baseline immune cell profiling data are also highlighted in Table 1. CrownBio research has shown that baseline immune cell populations in untreated syngeneic models (T cells and the T eff /T reg ratio) may predict efficacy of anti-ctla-4 and anti-pd-l1 antibodies, respectively (1). Example FACS analysis baseline data are included within Figure 1 for T cells (CD3 +, CD4 +, CD8 +, CD4 + /FOXP3 + ) and ratio of CD8 + T effect /T reg cells, with further NK, MDSC, and macrophage data available in MuBase.

4 Figure 1: Basal Level of Immune Cells in Syngeneic Tumors A: T cell mean % of CD3 +, CD4 +, CD8 +, CD4 + /FOXP3 + in TIL. B: Ratio of CD8 + T effect cells to T reg cells in total cell. Data generated at Crown Bioscience Taicang site. Mean (%) Fold Change Renca EL4 Renca EL4 MBT-2 MBT-2 LL/2 LL/2 ortho 4T1 ortho 4T1 CT-26.WT CT-26.WT B16-BL6 B16-BL6 RM-1 RM-1 H22 H22 L121 L121 B16-F1 B16-F1 EMT6 EMT6 A2 CD3 + CD3 + CD4 + CD3 + CD8 + T reg Table 1 also shows the availability of RNAseq data for our syngeneic models, which has been used to identify biomarkers to predict treatment response. Through generating detailed expression maps and mutational profiles, we have identified alternative gene splicing transcripts and gene fusions within our models. Further mutational analysis has indicated a number of our syngeneic models harbor mutations that may be useful for combination studies of targeted agents and immunotherapy, and we have identified a set of biomarkers that may be useful to predict immunotherapeutic agent response (2). We also provide syngeneic tumor samples for ex vivo research uses including tumor tissue histology (H&E staining), and frozen and formalin-fixed, paraffin-embedded tumor samples as required. Advanced Orthotopic and Metastatic Disease Models, and Syngeneic Imaging Modalities CrownBio also provides advanced syngeneic modeling options (model availability detailed in Table 1): orthotopic models to more closely recapitulate the tumor situation and microenvironment clinically relevant metastatic models of disease bioluminescent metastatic models to study clinically relevant metastatic invasion, metastatic lesions in secondary organs, and the evaluation of agents to target this metastasis. A2 Pan2 Pan2 P388D1 P388D1 L5178-R For more information on these models and our pipeline of developing bioluminescent syngeneics please request our Optical Imaging FactSheet. Examine a Vast Array of Checkpoint Inhibitor Benchmarking Data including Anti-PD1, PD-L1, and CTLA-4 Agents As checkpoint inhibitors continue to be approved for a variety of cancer types, preclinical evaluation via syngeneic models can be used to identify their potential indications and combination therapy strategies. CrownBio has extensively profiled our syngeneic panel in vivo response to a variety of checkpoint inhibitors, providing clients with the information necessary to select models and the correct doses for combination therapy (available data shown in Table 1). Waterfall plots for our models tested with anti-pd-1, anti-pd-l1, and anti- CTLA-4 antibodies are shown in Figure 2 through Figure 4. Figure 2: Anti-PD-1 Antibody Efficacy Benchmarking in Syngeneic Models Antibody: RMP1-14. All data mean + SD. Data generated at Crown Bioscience Beijing and Taicang sites. TGI (%) Hepa 1-6 1mg/kg H22 1mg/kg EM T6 5mg/kg ortho 4T1 1mg/kg L5178-R 5mg/kg CT26.WT 1mg/kg MBT- 2 5mg/kg L121 1mg/kg E.G7-OVA 2ug/mouse A2 1mg/kg subq4t1 1mg/kg LL /2 1mg/kg B16-F1 1mg/kg B16-BL 6 1mg/kg KLN25 5mg/kg RM -1 1mg/kg Pan2 5mg/kg P388D1 1mg/kg EL 4 1mg/kg MPC- 11 5mg/kg WEHI-164 5mg/kg Renca 1mg/kg Colon26 5mg/kg Figure 3: Anti-PD-L1 Antibody Efficacy Benchmarking in Syngeneic Models Antibody: 1F.9G2. All data mean + SD. Data generated at Crown Bioscience Beijing and Taicang sites. TGI (%) Hepa 1-6 5mg/kg CT26.WT 1mg/kg A2 1mg/kg EM T6 1mg/kg H22 5mg/kg L5178-R 5mg/kg CloudmanS91 5mg/kg RM -1 1mg/kg MBT- 2 5mg/kg P815 5mg/kg ortho 4T1 5mg/kg Renca 5mg/kg P388D1 5mg/kg EL 4 5mg/kg B16-F1 1mg/kg Pan2 5mg/kg subq4t-1 5mg/kg MP C- 11 5mg/kg L121 1mg/kg KLN25 5mg/kg LL /2 1mg/kg Colon26 5mg/kg B16-BL 6 5mg/kg

5 Figure 4: Anti-CTLA-4 Antibody Efficacy Benchmarking in Syngeneic Models A: 9D9, data mean + SD; B: 9H1. Data generated at Crown Bioscience Beijing and Taicang sites. A. B B. Tumor Voume (mm 3 ) IgG2a 3mg/kg b.i.w. TGI (%) TGI (%) Individual control and treated spider plots are available for each model on request, to evaluate model response variability, example data for the liver syngeneic Hepa 1-6 model is included in Figure 5. Figure 5: Variability of Hepa 1-6 Response: Control and Treatment Spider Plots A: Mean tumor volume ± SEM. B-E: Individual reponse following treatment with IgG2a or checkpoint inhibitor shown. Statistical analysis on Day 25 post inoculation. Data generated at Crown Bioscience Taicang site. H22 1mg/kg EM T6 1mg/kg Hepa 1-6 5mg/kg subq4t-1 1mg/kg MBT-2 1mg/kg CT26.WT 1mg/kg ortho 4T1 1 mg/kg RM -1 1mg/kg Pan2 1mg/kg A2 1mg/kg L121 1mg/kg B1 6-F1 5mg/kg B16-BL 6 1mg/kg LL /2 1mg/kg P388D1 1mg/kg Colon26 5mg/kg EL4 1mg/kg Renca 1mg/kg IgG2a 3mg/kg b.i.w. Anti-PD-1 (RMP1-14) 1mg/kg b.i.w. Anti-PD-L1 (1F.9G2) 5mg/kg b.i.w. Anti-CTLA-4 (9D9) 5mg/kg b.i.w Treatment T/C (%) TGI (%) p Value Anti-PD-1 (RMP1-14) <.1 Anti-PD-L1 (1F.9G2) Anti-CTLA-4 (9D9) <.1 L5178-R 1mg/kg MPC-11 1mg/kg KLN25 1mg/kg C. D. E Anti-PD-1 (RMP1-14) 1mg/kg b.i.w Anti-PD-L1 (1F.9G2) 5mg/kg b.i.w Anti-CTLA-4 (9D9) 5mg/kg b.i.w

6 Evaluate Combination Checkpoint Inhibitor and Chemotherapy Regimens As researchers discover that chemotherapy, radiotherapy, and targeted therapies may interact or change the tumor immune environment, suitable models are required to evaluate combinations of these agents with immunotherapy. CrownBio is utilizing our syngeneic panel to investigate combination therapy strategies. Example data treating the H22 liver cancer syngeneic model with a combination of doxorubicin and anti-pd-l1 antibody showed that combined treatment had a greater effect than either treatment alone (Figure 6). A range of checkpoint inhibitors have been trialed in combination with cyclophosphamide on the A2 B lymphoma model (Figure 7). Response to combination therapy varied, with the greatest tumor growth inhibition observed for cyclophosphamide combined with anti-gitr antibody. Figure 6: Combination Doxorubicin and Anti-PD-L1 Antibody Induces TGI of H22 Model Greater than Either Agent Alone Data generated at Crown Bioscience Taicang site Treatment 8 4 Vehicle q.w. Doxorubicin 5mg/kg q.4.d. Anti-PD-L1 (1F.9G2) 5mg/kg b.i.w. Doxorubicin 5mg/kg q.4.d. + Anti-PD-L1 (1F.9G2) 5mg/kg b.i.w Tumor Volume (mm 3 ) T/C Value (%) on Day 21 Vehicle 2291 ± p Value Anti-PD-L1 (1F.9G2) 519 ± <.1 Anti-CTLA-4 (9D9) 1436 ± Anti-PD-L1 (1F.9G2) 268 ± <.1 Figure 7: Cyclophosphamide and Checkpoint Inhibitor Combined Treatment of A2 Model Elicits a Range of Responses Data generated at Crown Bioscience Taicang site. Treatment PBS CTX 2mg/kg CTX 2mg/kg + Anti-CTLA-4 (9D9) 75μg/mouse CTX 2mg/kg + Anti-PD-1 (RPM1-14) 75μg/mouse CTX 2mg/kg + Anti-OX4 (OX-86) 75μg/mouse CTX 2mg/kg + Anti-GITR (DTA-1) 75μg/mouse CTX 2mg/kg + Anti-CD2 (AISB12) 5μg/mouse Tumor Volume (mm 3 ) T/C Value (%) on Day 24 p Value PBS 2752 ± Cyclophosphamide (CTX) 776 ± <.1 CTX + Anti-CTLA-4 (9D9) 61 ± <.1 CTX + Anti-PD1 (RMP1-14) 116 ± CTX + Anti-OX4 (OX-86) 538 ± CTX + Anti-GITR (DTA-1) 151 ± 91 5 <.1 CTX + Anti-CD2 (AISB12) 895 ± <.1 Combining Inducers of Immunogenic Cell Deacth (ICD) with Immunotherapy A number of anticancer treatment strategies such as chemotherapeutic agents (e.g. oxaliplatin, doxorubicin, bortezomib, and mitoxantrone), radiotherapy, and oncolytic viruses have been highlighted as potential inducers of ICD. These treatments are known to increase the presentation of cell-associated antigens to CD4 + and CD8 + T lymphocytes by dendritic cells. Combination strategies of ICDs with immunotherapies could therefore provide opportunities to harness the immune system to extend survival, even among metastatic and heavily pretreated cancer patients, and may increase the efficacy of immunotherapy in cancer types with low immunogenic status.

7 CrownBio has combined the ICD oxaliplatin with anti-ctla-4 in treating the CT26 colon cancer syngeneic model. Combination of anti-ctla-4 immunotherapy with oxaliplatin resulted in an additive tumor growth inhibition (Figure 8), and also induced a statistically significant increase in CD8 + TILs compared with oxaliplatin or anti-ctla-4 antibody alone (p<.5, Figure 9) (3). These results effectively demonstrate the applicability for further exploring combination ICD inducer strategies involving immunotherapy. Figure 8: ICD Oxaliplatin and Anti-CTLA-4 Combination Results in Additive TGI TGI reduction: Anti-CTLA-4 vs vehicle p<.5. Oxaliplatin vs vehicle p<.1. Combination therapy is additive over single agent therapy alone. Data generated at CrownBio UK Vehicle b.i.w. + vehicle q.w. Anti-CTLA-4 1mg/kg b.i.w. ( ) + vehicle q.w. Oxaliplatin 6mg/kg q.w. ( )+ vehicle q.w. Anti-CTLA-4 1mg/kg b.i.w. ( )+ oxaliplatin 6mg/kg q.w. ( ) Study Day Figure 9: ICD Oxaliplatin and Anti-CTLA-4 Combination Results in CD8+ TIL Increase Treatment dosing and regimens as per Figure 8. A: % CD3 + /CD8 + T cells, *p<.5 vs single agent anti-ctla-4 and oxaliplatin. B: Ratio T eff :T reg. Data generated at CrownBio UK. A. CD3 + CD8 + /Viable Single Lymphocytes Vehicle Anti-CTLA-4 Oxaliplatin Anti-CTLA-4 + Oxaliplatin * Figure 9: Continuation.. B. Ratio of CD8 + : CD4 + /FoxP Vehicle Anti-CTLA-4 Oxaliplatin Anti-CTLA-4 + Oxaliplatin Treatment Microbiome Analysis of Responder vs Non-Responder Animals Microbiota play an important role in determining an organism s response to anticancer treatment, even in tumors far from the gastrointestinal tract, possibly because of their pro-inflammatory properties which activate the immune system. In order to gain insights into the complex interaction between the microbiome and cancer therapy, CrownBio performs fecal collection and microbiome profiling (16S rrna sequencing) to compare gut microbiomes across our syngeneic models, which we can correlate with response to therapy. Example data is shown in Figure 1 for animals implanted with either CT-26 or 4T1 models, and treated with anti-pd-1 antibody or isotype control. Efficacy studies revealed varying response across different tumor models and within tumor models. Gut microbiome sequencing was performed post dosing and showed that: the gut microbiome of animals that were responsive to anti-pd-1 treatment differed from animals that were treated with isotype control the gut microbiome of animals that were unresponsive to anti-pd-1 treatment clustered closely with animals that were treated with isotype control (Figure 1) (4). Treatment

8 izing Syngeneic the Effect Models of Immune Factsheet Checkpoint Inhibitors on Syn Through Gut Microbiome Sequencing and Immunoph Figure 1: Gut Microbiome Variation between Responder vs Non-Responder Animals Efficacy studies: n=8; mean ± SEM. Gut microbiome examined by r16s sequencing of fecal samples collected post last dose of apd-1 or isotype control. In-between sample difference analyzed using pairwise comparisons Jayant of beta-diversity Thatte, by unweighted Tommy unifrac Broudy metric as displayed by Principal Component Analysis. Taxa abundance at the genus level is represented in stacked columns. Data generated at CrownBio San Diego. o Maves, Hooman Izadi, Elvira Catherina Talaoc, Andrew Calinisan, Deborah Yan, Charlene Echegara d the mune tment, rough er to TLA-4 typing mined shifts crossferent ations ments abled vidual odels trating ibitor Tumor Volume Tumor (mm Volume 3 ) (mm 3 ) T u m o r v o lu m e (m m 3 ) 3 3 DATA CT26 CT26 CT26 Anti-PD-1 Antibody 1mg/kg 3 2 Anti-PD-1 Antibody 2 1mg/kg 1 2 T u m o r v o lu m e (m m 3 ) T re a tm e n t d a y s 3 Treatment Days 1 Anti-PD-1 Antibody 1mg/kg Crown Bioscience Inc., San Diego, CA, USA T re a tm e n t d a y s Fig. 1. Fig. Immune 1. Immune Checkpoint Checkpoint Inhibitor Inhibitor Efficacy Efficacy Studies. Studies. In vivo In growth vivo growth characteristic characteristic of CT26 of and CT26 4T1 and syngeneic 4T1 syngeneic tumor tumor cell lines cell in lines subcutaneous in subcutaneous models. models. Eight animals Eight animals in each in group each were group treated were treated with apd-1 with apd-1 (n), or (n), respective or respective isotype isotype controls controls (l) after (l) randomization. after randomization. Animals were dosed at 1mg/kg on the indicated treatment days (arrows). Treatment Animals were Days dosed at 1mg/kg on the indicated treatment days (arrows). Treatment Data are Data displayed are Days displayed as as mean ± mean SEM. ± The SEM. inlaid The graph inlaid displays graph displays each curve each as curve measurements measurements from an from individual an individual animal. animal CT26 CT26 4T1 4T1 Treatment Days 1 2 T u m o r v o lu m e (m m 3 ) T u m o r v o lu m e (m m 3 ) T1 25 4T1 4T1 Anti-PD-1 Antibody 1mg/kg 2 15 Anti-PD-1 Antibody 1mg/kg T re a tm e n t d a y s Treatment Days T re a tm e n t d a y s Anti-PD-1 Antibody 1mg/kg Treatment Days Fig. 3. Fig. Immu 3 lymphocytes populations ( included. includ cience Isotype Control apd-1 apd-1 Isotype Control apd-1 apd-1 Fig. 2. Fig. Microbiome 2. Microbiome Sequencing. Sequencing. Gut microbiome Gut microbiome was examined was examined by r16sbysequencing sequencing of fecal ofsamples fecal samples collected collected post last post last dose of dose apd-1 of apd-1 (l) or (l) isotype or isotype control control (l). In-between (l). In-between sample sample difference difference was analyzed was analyzed using pairwise using pairwise comparisons comparisons of betadiversitdiversity by unweighted by unweighted unifrac unifrac metric metric as displayed as displayed by Principal by Principal Component Component Analysis. Analysis. Taxa abundance Taxa abundance at the genus at the genus level islevel is of beta- represented represented in stacked in stacked columns. columns. Re diff Gu tre con unr tha Imm cel ACKNO Many Many than sequencing Crown Crown Bios

9 Assess Immunotherapy Induced T Cell Infiltration and Immunomodulatory Effects Following checkpoint inhibitor or immunotherapy evaluation, CrownBio can perform immune cell profiling to evalute induced T cell infiltration and immuno-modulatory effects. Our techniques include FACS and IHC immunophenotyping, which have been validated with a range of our syngeneic models following checkpoint inhibitor treatment: Example FACS immunophenotyping data for the H22 liver model, and IHC immunophenotyping for the A2 lymphoma model are detailed below. The H22 model was treated with anti-pd-1 and anti-ctla-4 antibodies, with response to treatment correlating with an increase in selected tumor infiltrating lymphocytes (TIL) (Figure 11 and Figure 12). T cell infiltration into A2 tumors was analyzed via IHC and immunofluorescence (Figure 13). FACS immunophenotyping: MBT-2, 4T1, EMT6, CT-26.WT, L121, H22,B16-F1, and Pan2 models IHC immunophenotyping: A2 Figure 11: H22 Liver Syngeneic Model Responds to Checkpoint Inhibitors: Mean and Individual Response T/C values on Day 21: anti-pd-1 (RMP1-14) 16% (p=.2); anti-ctla-4 (9D9) 5% (p=.12). B, C, D: Individual responses to PBS control, anti-pd-1, and anti-ctla-4, respectively. Data generated at Crown Bioscience Taicang site PBS b.i.w. Anti-PD-1 (RMP1-14) 1mg/kg b.i.w. Anti-CTLA-4 (9D9) 1mg/kg b.i.w PBS b.i.w. 3 Anti-PD-1 (RMP1-14) 1mg/kg b.i.w. 3 Anti-CTLA-4 (9D9) 1mg/kg b.i.w

10 Figure 12: H22 Liver Syngeneic Model: Response to Checkpoint Abs Correlates with an Increase in Selected TILs FACS result on Day 21: 2 days post the 5th dose. Data generated at Crown Bioscience Taicang site. *p<.5, **p<.1, ***p<.1. Tumor Infiltrating Immune Cells (% in CD45 + Population) PBS b.i.w., i.p. actla-4 1mg/kg b.i.w., i.p. *** *** *** *** *** * CD4 + FOXP3 + apd-1 1mg/kg b.i.w., i.p. *** * * Figure 13: A2 Tumor T-Cell Infiltration IHC (images 2x) of CD4, CD8, CD335, FOXP3, and neutrophils (Ly6G/C) was used to label helper T-cells, cytotoxic T cells, NK, T reg, and neutrophil cells. All IHC assays were run with BondRX Autostainer (Leica) and stained on 4µm FFPE sections of A2 without treatment. IF (image 4x) of CD4 (red) and FOXP3 (green) was stained on frozen sections of the A2 model to label T reg cells (run on Bond RX). DAPI (blue) is used to label the nucleus. CD4 CD8 CD335 p Value vs Control CD45 + CD3 + CD3 + CD4 + CD3 + CD8 + CD4 +- FOXP3 + NK MDSC Macrophage Anti-PD-1 1mg/kg.82 <.1 <.1 < Anti-CTLA-4 1mg/kg.179 <.1 <.1.46 < FOXP3 Neutrophil CD4 FOXP3 DAPI Standard of Care and Experimental Treatment Data also Available A range of SoC agents, experimental treatments, and combination chemotherapies have been trialed with our syngeneic models (results shown in Table 2). Table 2: Syngeneic Model Standard of Care and Experimental Treatment Data Day: days post-tumor inoculation. Cancer Type Syngeneic Model Treatment T/C (%) p Value Breast 4T1 Paclitaxel Day 27: Colon CT-26 VEGF-TRAP Day 15: 5.7 Cisplatin Day 2: 52.2 Oxaliplatin Day 23: 5 <.1 Liver H22 Doxorubicin Day 21: 23 <.1 Sorafenib Day 28: Lymphoma A2 Cyclophosphamide Day 2: 6.4 <.1 Melanoma B16-BL6 Cisplatin Day 35: Melanoma B16-F1 Cisplatin Day 28: 3.6 Pancreatic Pan2 Gemcitabine Day 21: 58 <.1 Gemcitabine + cisplatin Day 21: 4 <.1 Gemcitabine + paclitaxel Day 45: 33 <.1

11 Fast-Track the In Vivo Screening of Immunotherapy Compounds For immunotherapeutic agents, in vitro screening is not the optimum approach for evaluating PD effect and/or efficacy across multiple cancer types. However, as an alternative, large-scale, parallel, in vivo screening of syngeneic models can provide a cost effective approach. CrownBio are therefore utilizing our syngeneic platform to offer a unique large scale MuScreen to fast track immunotherapy treatment strategies, the first platform of its type. MuScreen can be used for both single agent and combination studies, reducing variability and improving screening efficiency. We provide a syngeneic efficacy screening panel and tumor microarrays to fit your research needs. For further information please consult the MuScreen FactSheet available from the CrownBio website: publications/factsheets/. Conclusions Immunotherapy research and agents such as anti-pd-1 antibodies are showing considerable success in oncology; providing both patient benefits and commercial success for the pharmaceutical industry. However, progress in the field is hindered through a lack of experimental immunotherapy models featuring a fully competent immune system. Syngeneic models (allografts derived from immortalized mouse cancer cell lines, which originated from the same inbred strain of mice) are a simple way to evaluate novel immunotherapy treatments through eliciting an immune response, in fully immunocompetent mice. CrownBio has validated a large panel of syngeneic models, covering a variety of cancer types, with a commitment to further extend this model selection. Alongside subcutaneous models, bioluminescent imaging of orthotopic and metastatic tumous allows more clinically relevant stromal interactions to be modeled and investigated. Full characterization including immunoprofiling, NGS, and checkpoint inhibitor benchmarking allows rapid selection of appropriate models for client studies. Immunomodulatory effects of novel agents can be evaluated through assessment of immunotherapy induced T cell infiltration, validated for a range of models. Our models are also available for a wide variety of agent assessment from checkpoint inhibitors to other immnunotherapeutics including bacterial, viral, and vaccination research. As immunotherapies are combined with chemotherapy and targeted agents, in an effort to extend patient survival, CrownBio is also utilizing its wide ranging Syngeneic Panel to interrogate different combinations including with inducers of ICD and can offer a large scale MuScreen to fast-track strategies. References 1 Zhang L, Zhang J, Guo S et al. RNAseq and immune profiling analysis of syngeneic mouse models treated with immune checkpoint inhibitors enable biomarker discovery and model selection for cancer immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 215 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Molecular Cancer Therapeutics 215;14(12 Suppl 2): Abstract nr A6. 2 Zhang L, Zhang J, Guo S et al. RNAseq and FACS profiling of syngeneic mouse models treated with immune checkpoint inhibitors enable biomarker discovery and model selection for cancer immunotherapy [abstract]. In: Proceedings of the 17th Annual Meeting of the American Association for Cancer Research; 216 Apr 16-2; New Orleans, LA. Philadelphia (PA): AACR; Cancer Research 216;76(14 Suppl): Abstract nr Mckenzie A, Kumari R, Shi Q et al. Immune competent syngeneic models demonstrate additive effects of combination strategies using checkpoint immunotherapy and inducers of immunogenic cell death (ICD). [abstract]. In: Proceedings of the 17th Annual Meeting of the American Association for Cancer Research; 216 Apr 16-2; New Orleans, LA. Philadelphia (PA): AACR; Cancer Research 216;76(14 Suppl): Abstract nr Kato Maves Y, Izadi H, Talaoc EC et al. Characterizing the effect of immune checkpoint inhibitors on syngeneic tumor models through gut microbiome sequencing and immunophenotyping [abstract].. In: Proceedings of the 28th EORTC-NCI-AACR Symposium: Molecular Targets and Cancer Therapeutics; 216 Nov 29 Dec 2; Munich, Germany: European Journal of Cancer 216;69: S111. Contact Sales US: UK: +44 () busdev@crownbio.com Schedule Scientific Consultation Request a consultation to discuss your project. consultation@crownbio.com Explore Scientific Data Log into MuBase to review syngeneic model data. mubase.crownbio.com Version 3.